PROJECT SUMMARY Human congenital heart defects (CHD) are very common, occurring in nearly 1% of live births. Moreover, cardiovascular (CV) failures are the leading cause of birth defect- related deaths in infants. It is well established that biomechanical stimuli are important regulators of CV development. Thus, defining how mechanical factors are integrated with genetic pathways to coordinate mammalian heart tube function and morphogenesis is critically important for understanding CHD and heart failure. Such information will also factor heavily into strategies for new therapeutic interventions to treat/prevent CHD. Toward that end, the mouse model is an excellent system in which to study human congenital defects. However, due to the internal nature of mammalian development, analysis of heart biomechanics is challenging. Through the previous cycle of this grant, we established a set of innovative optical coherence tomography (OCT) approaches for live, high-resolution 3D imaging and quantitative assessment of mouse embryo CV dynamics. These techniques were applied to analysis of the pumping mechanism of the E8.5 to E10.5 mouse heart and characterization of mutant phenotypes mimicking human CHDs. Therefore, we are in a unique position to investigate how mechanical stimuli of cardiodynamics and blood flow are linked to molecular/genetic changes during early cardiac differentiation in living mouse embryos. While multiple studies suggest that cardiac contraction, blood flow and stiffness each influence CV development, due to the interdependence of these factors, their individual roles are unknown. The goal of this proposal is to define the differential role of cardiac contraction and flow-induced shear stress in regulating mechanical homeostasis (stiffness) and cell fate decisions in vivo. These experiments will specifically address the context-dependent interplay between these factors, which likely vary between cardiac regions with different functional roles, such as in actively contracting regions versus the passively contracting outflow tract (OFT). Scientific Premise, Scientific Rigor, and Relevant Biological Variables: This proposal will fill a significant gap in the field of early mammalian cardiac development and define the role of cardiac forces in maintaining mechanical homeostasis and cell differentiation. This information will lead to a better understanding, prevention and treatment of CHD and embryonic cardiac failures in humans. The proposed study is supported by strong preliminary data. We carefully articulated the number of experimental animals to be used, the precise genetic makeup of these animals, and the rationale for the choice of the models. Sex as a biological variable is considered and addressed in the proposal. Extensive details are provided to ensure that preliminary and proposed experiments can be replicated in other laboratories.